BIOCHEMISTRY OF ENERGY DEPENDENT (INTRACELLULAR) PROTEIN DEGRADATION

我们的研究集中在结构/功能关系的 ATP依赖性Clp和Lon蛋白酶，其降解重要的调节蛋白， 蛋白以及E.大肠杆菌和人 细胞野生型Lon可以使用牛痘表达载体来表达。 系统，蛋白质被靶向线粒体并被加工。 人类Lon的加工似乎是自催化的，因为突变体 其中活性位点丝氨酸残基已被改变的Lon被靶向 线粒体，但不加工和共表达的突变体， 野生型Lon蛋白酶导致突变体的加工。 野生型Lon在HeLa细胞中的表达导致细胞变圆 并失去活力，而丝氨酸突变体的表达没有 有害影响。该系统将被用来定义功能 不同Lon突变体的变化，并确定生理靶点 人类Lon蛋白酶的合成人Lon的稳定转染子已经被证实是 使用具有MDR 1作为显性选择标记的构建体获得。 在该系统中，失活突变体的高水平表达， 而不是野生型Lon。我们克隆并表达了E. coliClpX是ATP酶Clp家族的成员。ClpX具有ATP依赖性 分子伴侣的活性，并需要一些特定的ATP依赖 依赖于ClpP的蛋白水解活性。凝胶过滤和电子 显微镜显示，ClpX亚基（Mr 46，000）结合形成一个六- 一元环（Mr 280，000），通过与ATP结合而稳定。在 在ATP存在下，六聚体ClpX与十四聚体ClpP相互作用， 由叠加的七元环组成，形成稳定的复合物 其可以通过凝胶过滤分离。在复合体中， ClpP在每一侧上由ClpX的单环侧接。的对称 因此，在ClpP的七元环和ClpP的七元环之间存在错配。 ClpXP和ClpAP的六元ATP酶环。竞争研究 表明ClpX和ClpA具有几乎相等的结合亲和力， ClpP，但没有证据表明ClpA、ClpX和ClpP的混合复合物 表明一个特定的ATP酶与一个表面的结合 ClpP的表面有利于类似的ATP酶与ClpP的相对表面结合。 寡肽FAPHMALVPV在存在下被ClpXP切割， 转换数为10，000 min-1的ATP的不可水解类似物 (per ClpP的十四聚体），表明ClpX，如同ClpA， 变构激活影响ClpP，使活性位点更多 并增强蛋白水解的催化效率 活性部位ClpP中亚基相互作用的研究表明， 环之间的接触可以被破坏而不破坏子单元 七聚体环内的相互作用。高盐处理 浓度和低温导致可逆的分离， ClpP环，它可以重新与酶的完全恢复， 活动

Our research is focused on the structure/function relationships of the ATP-dependent Clp and Lon proteases, which degrade important regulatory proteins as well as damaged and denatured proteins in E. coli and human cells. Wild-type Lon can be expressed using a Vaccinia expression system, and the protein is targeted to mitochondria and processed. Processing of human Lon appears to be autocatalytic, because a mutant Lon in which the active site serine residue has been altered is targeted to mitochondria but is not processed and co-expression of the mutant and the wild-type Lon proteases leads to processing of the mutant. Expression of wild-type Lon in HeLa cells causes the cells to round up and lose viability, whereas expression of the serine mutant has no deleterious effects. This system will be exploited to define functional changes in different Lon mutants and to identify physiological targets of human Lon protease. Stable transfectants of human Lon have been obtained using a construct with MDR1 as the dominant selectable marker. In this system also, high level expression of the inactive mutant but not of wild-type Lon has been possible. We have cloned and expressed E. coli ClpX, a member of the Clp family of ATPases. ClpX has ATP-dependent chaperone activity and is required for some specific ATP-dependent proteolytic activities dependent on ClpP. Gel filtration and electron microscopy show that ClpX subunits (Mr 46,000) associate to form a six- membered ring (Mr 280,000) that is stabilized by binding of ATP. In the presence of ATP, hexameric ClpX interacts with ClpP, a tetradecamer composed of superimposed seven-membered rings, to form a stable complex that can be isolated by gel filtration. In the complex, the rings of ClpP are flanked on each side by a single ring of ClpX. A symmetry mismatch thus exists between the seven-membered rings of ClpP and the six-membered ATPase rings for both ClpXP and ClpAP. Competition studies showed that ClpX and ClpA have nearly equal affinity for binding to ClpP, however no evidence for mixed complexes of ClpA, ClpX, and ClpP were observed suggesting that binding of a specific ATPase to one face of ClpP favors binding of a like ATPase to the opposite face of ClpP. The oligopeptide, FAPHMALVPV, is cleaved by ClpXP in the presence of non- hydrolyzable analogs of ATP with a turnover number of 10,000 min-1 (per tetradecamer of ClpP), indicating that ClpX, as does ClpA, allosterically activates affects ClpP to make the active site more accessible and to potentiate the catalytic efficiency of the proteolytic active site. Studies of subunit interactions in ClpP indicate that contacts between rings can be disrupted without breaking the subunit interactions within the heptameric rings. Treatment with high salt concentrations and low temperature lead to reversible separation of the ClpP rings, which can reassociate with complete restoration of enzymatic activity.